Evaluation of antibody variants using a ribosome display and Brevibacillus choshinensis secretion system

In antibody engineering, the development of rapid and efficient strategies for improving affinity is highly necessary. In this study, we aimed to establish a method to efficiently enrich and analyze high-affinity antibody variants by combining protein synthesis using recombinant elements (PURE) ribosome display with next-generation sequencing (NGS) and Brevibacillus choshinensis secretion system using the NZ-1 antibody, which targets the PA tag peptide (GVAMPGAEDDVV) as a model antibody. From the mutated scFab library designed based on the structure, we performed a single-round of PURE ribosome display selection and analyzed the data by NGS to obtain high-affinity scFab candidates with high enrichment factor and high read counts. Subsequently, the most promising candidate was produced as a Fab in the B. choshinensis secretion system, and the purified Fab had an affinity (K_D = 1.6 × 10^-9 M) similar to the wild type. Overall, this study highlights the potential of the integrated PURE ribosome display with NGS analysis and the B. choshinensis secretion system for the rapid identification and analysis of high-affinity antibody variants.